Supplementary Figure 3: Screening for an optimal fluorescent dye and conjugation site on the tIVR sensor.
From: High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination
a, Cys residue in the linker connecting IsoTBuz and Vps27UIM; b, N46C; and c, M105C in IsoTBuz were selected to conjugate various fluorescent dyes. In each panel, the arrow shows the conjugation site on the tIVR model and the histograms show performance with different fluorophores installed at that site. To determine which site and fluorescent dye gave the largest fluorescence change upon Ub binding, fluorescence intensities of the labeled sensors (2 nM) were measured with and without saturating Ub. F0 is fluorescence intensity of the labeled sensors without Ub, and ∆F is the intensity change upon addition of 200 µM Ub.
